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Theriogenology ; 132: 95-105, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004879


The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.

Blastocisto/fisiologia , Desenvolvimento Embrionário/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Técnicas de Silenciamento de Genes/veterinária , Partenogênese , Suínos/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Suínos/genética
Reprod Fertil Dev ; 31(3): 473-481, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30301509


Faithful repair of DNA double-strand breaks in mammalian oocytes is essential for meiotic maturation and embryonic development. In the present study we investigated the roles of Roscovitine and Trichostatin A (TSA) in DNA damage recovery during invitro maturation of porcine oocytes. Etoposide was used to trigger DNA damage in oocytes. When these DNA-damaged oocytes were treated with 2µM Roscovitine, 50nM TSA or both for 22h, first polar body extrusion and blastocyst formation in all treated groups were significantly improved compared with the etoposide-only group. The most significant improvement was observed when Roscovitine was present. Further immunofluorescent analysis of γH2A.X, an indicator of DNA damage, indicated that DNA damage was significantly decreased in all treated groups. This observation was further supported by analysing the relative mRNA abundance of DNA repair-related genes, including meiotic recombination 11 homolog A (MRE11A), breast cancer type 1 susceptibility protein (BRCA1), Recombinant DNA Repair Protein 51 (RAD51), DNA-dependent protein kinase catalytic subunit (PRKDC) and X-ray cross complementing gene 4 (XRCC4). Compared with the etoposide-only group, the experimental group with combined treatment of Roscovitine and TSA showed a significant decrease of all genes at germinal vesicle and MII stages. The Roscovitine-only treatment group revealed a similar tendency. Together, these results suggest that Roscovitine and TSA treatments could increase the capacity of oocytes to recover from DNA damage by enlisting DNA repair processes.

Reparo do DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Roscovitina/farmacologia , Animais , Feminino , Técnicas de Maturação in Vitro de Oócitos , Suínos
Cryobiology ; 69(1): 55-60, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24854867


INTRODUCTION: We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing-thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. MATERIAL AND METHODS: H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen-thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. RESULTS: The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05). CONCLUSIONS: This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles.

Proteína Quinase CDC2/genética , Cadeias Pesadas de Clatrina/genética , Criopreservação , DNA Topoisomerases Tipo I/genética , Histonas/genética , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , DNA Complementar/genética , Fertilidade , Fertilização , Congelamento/efeitos adversos , Oócitos/citologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
Reprod Biol Endocrinol ; 12: 30, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24754924


BACKGROUND: The competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF. METHODS: In Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen-thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 µg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0-6 h or 18-24 h after initiation of maturation. RESULTS: The optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P<0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P<0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM. CONCLUSIONS: An optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF.

Hormônio Foliculoestimulante/administração & dosagem , Leupeptinas/administração & dosagem , Hormônio Luteinizante/administração & dosagem , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Inibidores de Proteassoma/administração & dosagem , Animais , Bovinos , Células Cultivadas , Meios de Cultura , Feminino
Cryobiology ; 68(1): 152-4, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24463092


In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.

Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Fertilização/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Dimetil Sulfóxido/farmacologia , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Etilenoglicol/farmacologia , Feminino , Fertilização/fisiologia , Fertilização In Vitro , Ficoll/farmacologia , Masculino , Oócitos/citologia , Oócitos/fisiologia , Concentração Osmolar , Sacarose/farmacologia , Vitrificação